Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg²⁺ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg²⁺ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.     

Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes

The complete VS ribozyme comprises seven helical segments, connected by three three-way RNA junctions. In the presence of Mg²⁺ ions, cleavage occurs within the internal loop of helix I. This requires the participation of a guanine (G638) within the helix I loop, and a remote adenine (A756) within an internal loop of helix VI. Previous structural studies have suggested that helix I docks into the fold of the remaining part of the ribozyme, bringing A756 and G638 close to the scissile phosphate to allow the cleavage reaction to proceed. We show here that while either A756C or G638A individually exhibit very low cleavage activity, a mixture of the two variants leads to cleavage of the A756C RNA, but not the G638A RNA. The rate of cleavage depends on the concentration of the VS G638A RNA, as expected for a bimolecular interaction. This regaining of cleavage activity by complementation indicates that helix I of one VS RNA can interact with another VS RNA molecule to generate a functional active site in trans.     

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.     

Concerted folding of a Candida ribozyme into the catalytically active structure posterior to a rapid RNA compaction

Folding of the major population of Tetrahymena intron RNA into the catalytically active structure is trapped in a slow pathway. In this report, folding of Candida albicans intron was investigated using the trans-acting Ca.L-11 ribozyme as a model. We demonstrated that both the catalytic activity (k_obs) and compact folding equilibrium of Ca.L-11 are strongly dependent on Mg²⁺ at physiological concentrations, with both showing an Mg²⁺ Hill coefficient of 3. Formation of the compact structure of Ca.L-11 is shown to occur very rapidly, on a subsecond time scale similar to that of RNase T1 cleavage. Most of the ribozyme RNA population folds into the catalytically active structure with a rate constant of 2 min^–1 at 10 mM Mg²⁺; neither slower kinetics nor obvious Mg²⁺ inhibition is observed. These results suggest that folding of the Ca.L-11 ribozyme is initiated by a rapid magnesium-dependent RNA compaction, which is followed by a slower searching for the native contacts to form the catalytically active structure without interference from the long-lived trapped states. This model thus provides an ideal system to address a range of interesting aspects of RNA folding, such as conformational searching, ion binding and the role of productive intermediates.     

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg²⁺ ion concentrations are low, K⁺ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg²⁺ (0.5–2 mM) and K⁺ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.     

Biochemical analysis of pistol self-cleaving ribozymes

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min⁻¹ under physiological Mg²⁺ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2′-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.     

Biochemical analysis of hatchet self-cleaving ribozymes

Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg²⁺ to promote RNA strand scission with a maximum rate constant of ∼4 min⁻¹. As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2′-oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.     

Molecular beacons as probes of RNA unfolding under native conditions

Hybridization of fluorescent molecular beacons provides real-time detection of RNA secondary structure with high specificity. We used molecular beacons to measure folding and unfolding rates of the Tetrahymena group I ribozyme under native conditions. A molecular beacon targeted against 15 nt in the 5′ strand of the P3 helix specifically hybridized with misfolded forms of the ribozyme, without invading the native tertiary structure. The beacon associated with the misfolded ribozyme 300 times more slowly than with an unstructured oligonucleotide containing the same target sequence, suggesting that the misfolded ribozyme core remains structured in the absence of Mg²⁺. The rate of beacon hybridization under native conditions revealed a linear relationship between the free energy of unfolding and Mg²⁺ concentration. A small fraction of the RNA population unfolded very rapidly, suggesting parallel unfolding in one step or through misfolded intermediates.     

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg²⁺ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg²⁺ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg²⁺ or Ca²⁺ concentration.     

Interaction between RNA molecules of a two-component trans analog of antigenomic HDV ribozyme

A study was made of the association of the RNA components forming a B:LS two-component rans analog of the antigenomic HDV ribozyme. The B:LS ribozyme differed from known trans ribozymes in the sizes and nucleotide sequences of its components (33 and 34 nt, respectively), the topology of its functional parts, and the lack of a very short cleavage product. Compared to the cis ribozyme, B:LS showed similar dependences on the reaction conditions (Mg²⁺ concentration, pH, temperature) and a similar biphasic kinetic curve of self-cleavage. The kinetic model of B:LS self-cleavage (available at www.cardio.ru/labgen/RZ_e.html) describes a possible cause of the biphasic kinetic curve as a change in the rate-limiting step of consecutive conformational transitions accompanying self-cleavage. Another possible cause is an interaction between the molecules involved in cleavage, i.e., multimerization of whole ribozyme molecules with their components or the reaction products. B:LS provides a convenient model for studying such interactions, since the mode of component binding allows generation of 1B:2LS and 2B:1LS complexes as well as complexes with the cleavage products. Nondenaturing PAGE was used to study the factors affecting association and dissociation of the ribozyme components. The possibility of interactions between the RNA components of the cis and trans ribozymes was demonstrated experimentally. It was shown that the ribozyme is capable of multimerization when LS is in excess over B and that the cleavage products are not significantly involved in this process. The results suggest intermolecular interactions for the cleavage of the natural cis ribozyme.     

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3′-terminus. The variants show high cleavage activity in the presence of Mg²⁺, Ca²⁺ and Mn²⁺, lower with Co²⁺ and Sr²⁺ and some variants are also active with Cd²⁺ and Zn²⁺ ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg²⁺ is partially inhibited in the presence of Na⁺, spermidine and some other divalent metal ions. The inert Co(NH₃)₆³⁺ complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.     

Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2′-O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg²⁺ ion facilitates deprotonation of the O2′ nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5′ leaving group as noted in a previous study. This model assumes that the active site Mg²⁺ ion forms an inner-sphere coordination with the O2′ nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg²⁺ ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg²⁺ are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg²⁺ is simply replaced with Na⁺ is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg²⁺-bound starting state, which has not yet been definitively verified experimentally, nor explored in depth computationally. Thus, further experimental and theoretical study is needed such that a consensus view of the catalytic mechanism emerges.     

Threshold Occupancy and Specific Cation Binding Modes in the Hammerhead Ribozyme Active Site are Required for Active Conformation

The relationship between formation of active in-line attack conformations and monovalent (Na⁺) and divalent (Mg²⁺) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg²⁺ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg²⁺, in addition to stabilizing active conformations, plays a specific chemical role in catalysis.     

Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method

We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’ and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg²⁺, Ca²⁺ and Mn²⁺ ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis.     

Accommodation of Ca(II) ions for catalytic activity by a group I ribozyme

The wildtype Tetrahymena ribozyme cannot catalyze detectable levels of phosphotransfer activity in vitro on an exogenous RNA substrate oligonucleotide when calcium(II) is supplied as the only available divalent ion. Nevertheless, low-error mutants of this ribozyme have been acquired through directed evolution that do have activity in 10 mM CaCl₂. The mechanisms for such Ca(II) accommodation are not known. Here, we assayed the entire molecule in an effort to identify the roles of the mutations in allowing catalytic activity in Ca(II). We used four biochemical probing techniques - native-gel electrophoresis, hydroxyl radical footprinting, terbium(III) cleavage footprinting, and phosphorothioate interference mapping - to compare the solution structure of the wildtype ribozyme with that of a Ca(II)-active five-site mutant. We compared the gross folding patterns and specific metal-binding sites in both MgCl₂ and CaCl₂ solutions. We detected no large-scale folding differences between the two RNAs in either metal. However, we did discover a limited number of local folding differences, involving regions of the RNA affected by positions 42, 188, and 270. These data support the notion that Ca(II) is accommodated by the Tetrahymena ribozyme by a slight breathing at the active site, but that alterations at, near to, and distal from the active site can all contribute to Ca(II)-based activity.     

The mechanism of acidic hydrolysis of esters explains the HDV ribozyme activity

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme that catalyzes the site-specific trans-esterification reaction. Using high hydrostatic pressure (HHP) technique we showed that HDV ribozyme catalyzes the reaction of RNA cleavage in the absence of magnesium ions according to mechanism of acidic hydrolysis of esters. HHP induces changes of water structure, lowering pH and effect ribozyme catalytic site structure formation without magnesium. HHP, similarly to magnesium ion at ambient pressure stabilizes the higher order RNA structure of HDV, but Mg²⁺ is not involved in the catalysis. Our results clearly support the new mechanism of HDV hydrolysis and show advantages of using HHP in analysis of macromolecules interaction.     

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg²⁺ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg²⁺ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg²⁺ concentration that arises from association with several Mg²⁺ ions. This transition is followed by a second Mg²⁺-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ~100ms timescale offers insight into the folding dynamics of this ribozyme.     

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg²⁺, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg²⁺ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg²⁺-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg²⁺, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg²⁺ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg²⁺-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg²⁺-ion interactions contribute to RNA structural properties and stability.